A variety of strategies are available for the delivery of genes into cells. Transfection, in particular, refers to the delivery or transfer and uptake of nucleic acids into cultured cells mediated by gene transfer techniques. Commonly utilized transfection techniques generally tend to fall within two categories: transfection by physical methods (e.g., biolistic particle delivery and electroporation) and transfection by biochemical methods.
Biochemical methods of transfection have been used for a number of years to deliver nucleic acids into cultured cells. Such techniques may involve, for example, the use of inorganic aggregates (e.g., calcium-phosphate-mediated transfection), cationic polymers (e.g., diethylaminoethyl (DEAE)-dextran-, polylysine-, or polybrene-mediated transfection) and, more recently, cationic lipid (liposome) reagents (e.g., Lipofectamine™, Invitrogen, Carlsbad, Calif.). Other polycationic macromolecules and/or amphiphilic aggregates have also been developed which ionically condense nucleic acids and bind to the cell surface (see, e.g., Cotton et al., Curr. Opin. Biotechnol. (1993), 4, 705-710; Frese et al., Adv. Drug Delivery Rev. (1994), 14, 137-152; Haensler et al., Bioconjugate Chem. (1993), 4, 372-379; Behr et al., Bioconjugate Chem. (1994), 5, 382-389; Legendre et al., Proc. Natl. Acad. Sci. USA (1993), 90, 893-897). One particular polycationic composition, polyethyleneimine, has been shown to be a relatively efficient vehicle for delivering nucleic acids to cells both in vivo and in vitro (see, e.g., Boussif et al., Proc. Natl. Acad. Sci. USA (1995), 92, 7297-7301).
Polyethyleneimine is an organic macromolecule having a relatively high cationic charge density potential. Thus, it readily binds and condenses nucleic acids into small condensates which can be taken up (i.e., endocytosed) by cells. Additionally, every third atom of polyethyleneimine is an amino moiety that can be protonated; thus, the polymeric network can act as a “proton sponge” at a variety of pH's, using the hydrogen ion buffering polyamines to absorb hydrogen ions during the acidification of the endosome which leads to endosome lysis. See, e.g., Boussif et al., supra; Horbinski et al., BMC Neuroscience (2001), 2, 2.
The use of mammalian cell lines for recombinant gene expression provides a number of advantageous features such as proper folding and post-translational modification of the recombinant polypeptides. Chinese hamster ovary (CHO) cells, in particular, are a frequently used host cell for the production of recombinant polypeptides that require post-translational modification to express full biological function.
Derouazi et al. (Biotechnol. Bioeng. (2004), 87(4), 537-545) disclose cultivating CHO cells from the CHO-DG44 cell line in a serum-free, chemically defined medium and transfecting them using a transfection agent including linear or branched polyethyleneimine and DNA. In particular, Derouazi et al. disclose seeding the CHO cells in a 3 L bioreactor at a cell density of 2×106 cells/ml of cell culture medium. Immediately after seeding, the cells were transfected with the polyethyleneimine/DNA mixture, which was formed by diluting DNA and polyethyleneimine separately in 150 mM NaCl or 278 mM glucose and adding the polyethyleneimine dilution to the DNA dilution to form the transfection agent.
Although the above-described techniques and processes are generally useful for the transfection and expression of recombinant polypeptides in CHO cells, there are limitations on their effectiveness, including, for example, limited transfection efficiency and lower amounts of recombinant polypeptide expressed and/or produced by the transfected CHO cells, particularly in the large-scale culture and transfection of CHO cells. As a result of lower transfection efficiency, for instance, a lower amount of recombinant polypeptide may be expressed by the CHO cells. Accordingly, a need remains for additional processes for the transfection of CHO cells having improved transfection efficiencies and/or greater recombinant polypeptide production and/or expression by the transfected CHO cells.